Mice
This study was approved by the ethical committee for animal experiments in Gothenburg. Female C57BL/6J mice (Charles River Laboratories, Sulzfeld, Germany) were kept under standard environmental conditions and fed soy-free chow and tap water ad libitum. All mice in the experiment, both in the non-arthritic group (“control”, n = 14) and in the arthritic group (“CAIA”, n = 15), were ovariectomized at 8 weeks of age as described previously [12]. Successful removal of ovaries was confirmed by weighing uteri at termination of the experiment.
Arthritis induction
Two weeks after ovariectomy, the ArthritoMab™ CII mAb cocktail for C57BL/6 (8 mg/mouse; MD Biosciences GmbH, Zürich, Switzerland) was injected intravenously to induce arthritis (CAIA group). Non-arthritic control mice received phosphate-buffered saline. Three days after antibody administration, 100 μg LPS (Escherichia coli 055:B5; MD Biosciences) was injected intraperitoneally to CAIA and control mice. Mice were randomly assigned to experimental groups. The experiment was ended 9 days after antibody administration. This day for termination was chosen based on previous pilot studies showing that arthritis incidence peaked at day 6 after antibody administration and that arthritis severity decreased after day 7.
Arthritis evaluation
Arthritis incidence and severity were evaluated daily in a blinded manner. Severity was graded 0–3 in each paw (with a total maximum score of 12 per mouse) as follows: swelling in digits: 0.25 points per digit, maximum 1 point per paw; mild, intermediate, or severe swelling in metacarpal/tarsal joints: 0.5, 0.75, or 1 points, respectively; and mild, intermediate, or severe swelling in carpal/tarsal joints: 0.5, 0.75, or 1 points, respectively.
High-resolution micro-computed tomography
High-resolution micro-computed tomography (μCT) analyses were performed using an 1172 micro-CT model (Bruker, Aartselaar, Belgium) as described previously [12]. Trabecular bone parameters were analyzed in the distal metaphyseal region while the cortical bone parameters were analyzed in the diaphyseal region of femur [12].
Enzyme-linked immunosorbent assay
Sera were stored at −20 °C until use. Complement factor 3 (C3; Immunology Consultants Laboratory, Inc., Portland, OR, USA), cartilage oligomeric matrix protein (COMP; AnaMar AB, Gothenburg, Sweden), C-terminal telopeptides of type I collagen (CTX-I; Immunodiagnostics Systems Ltd, Boldon, UK), and N-terminal propeptide of type I procollagen (PINP; Immunodiagnostics Systems Ltd) were measured by enzyme-linked immunosorbent assay (ELISA) in serum diluted 1:50,000, 1:10, 1:2, and 1:1, respectively, according to the manufacturer’s instructions. The assay detection limits for C3, CTX-I, and PINP were 1.379 ng/ml, 2 ng/ml, and 7 ng/ml, respectively. The sensitivity of the COMP ELISA was 0.02 U/l.
Histomorphometry
Tartrate-resistant acid phosphatase activity was demonstrated in femurs as previously described by Toyosawa et al. [13]. The number of osteoclasts (tartrate-resistant acid phosphatase-positive nucleated cells on the bone surface) in the distal femoral epiphysis was counted and divided by the bone surface using a Nikon Eclipse 80i microscope with Osteomeasure™ software (v.3.2.1.7; Osteometrics Inc., Decatur, GA, USA).
Preparation of cells and flow cytometry analysis
Bone marrow was flushed from the femur using a syringe. Lymph nodes draining the joints were mashed in a 70 μm cell strainer. Single cell suspensions were stained for surface markers using anti-CD3 Horizon V500 and anti-CD11b Horizon V500 (Becton, Dickinson & Company (BD), Franklin Lakes, NJ, USA), anti-CD4 fluorescein isothiocyanate (FITC), anti-MCSF-R allophycocyanin (APC), and anti-F4/80 FITC (BioLegend, San Diego, CA, USA) antibodies. Lymphocytes were gated on singlet cells and CD4+ T cells were defined as CD3+CD4+ cells. Preosteoclasts were gated on singlet cells and defined as CD11b+F4/80+MCSF-R+ cells. For detection of interleukin (IL)-17+ cells in lymph nodes, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml; Sigma), ionomycin calcium salt (1 μg/ml; Sigma) and Golgiplug® (BD) for 4 hours at 37 °C and 5 % CO2, and stained intracellularly with anti-IL-17A APC (eBioscience, Vienna, Austria). Samples were run on a BD FACS Canto II and data were analyzed using the Flow Jo 8.8.6 or 10.0.6 software (Three Star Inc, Ashland, OR, USA).
Statistical analysis
Statistical analyses were performed using SPSS software 21.0 (IBM, Armonk, NY, USA). Student’s t test was used for comparison of two independent groups. Logarithmic transformations were used when appropriate to ensure normal distribution of data. Experiments were terminated on different days; variation between days was therefore assessed and corrected for when needed using analysis of covariance. The log-rank test was used to analyze arthritis incidence, and data are presented as Kaplan–Meier curves. The area under the curve for arthritis severity versus time was calculated for each mouse by the trapezoidal method:
$$ \mathrm{Area} \approx 0.5\left({y}_0+{y}_1\right)\Delta x + 0.5\left({y}_1+{y}_2\right)\Delta x + 0.5\left({y}_2+{y}_3\right)\Delta x + \dots $$
where ∆x is the time between arthritis assessment and y
0, y
1, y
2, y
3, etc. is the arthritis severity score for day 0, 1, 2, 3, etc. Since the scoring was performed using an ordinal scale, comparisons between groups were analyzed by non-parametric Mann–Whitney test. Differences in n are due to lack of sample, sickness, or laboratory errors. All tests are two sided. Data are presented as mean + standard error of the mean, unless otherwise stated. p <0.05 was considered significant.